The breast oncogene ErbB-2 is amplified or overexpressed in 15-30% of breast cancers. ErbB-2 overexpression is associated with resistance to cytotoxic and anti-hormone treatment and poor overall survival. Anti-ErbB-2 treatment strategies include the use of trastuzumab, a humanized monoclonal antibody directed against ErbB-2, plus a taxane-based chemotherapy or lapatinib, a dual EGFR and ErbB-2 tyrosine kinase inhibitor-approved for trastuzumab resistant disease. Unfortunately, resistance to trastuzumab and now lapatinib is a major problem in metastatic breast cancer with only 30-50% responding to anti-ErbB-2 therapy. Therefore, the identification of compensatory survival pathways that contribute to anti-ErbB-2 treatment resistance is important in order to design rational targeted treatment options to prevent or reverse resistance and improve overall survival. We have identified that Notch-1, another potent breast oncogene and cell fate determinant, is activated by trastuzumab or a TKI similar to lapatinib in ErbB-2 positive breast cancer cells and served as a novel therapeutic target for anti-ErbB-2 treatment resistance (1). Our findings suggested that there is a compensatory increase in Notch-1-mediated proliferation/survival in trastuzumab or a TKI similar to lapatinib-treated breast cancer (1). The Notch-1 receptor is another potent breast oncogene (2) that is overexpressed with its ligand Jagged-1 in breast cancers associated with the poorest overall survival (3-5). Furthermore, Notch has been shown to be necessary for the proliferation and survival of breast cancer stem cells (6). Therefore, it i critical to identify the mechanism(s) by which ErbB-2 inhibition activates Notch-1 activity and whether suppression of the Notch pathway prevents and/or reverses resistance to trastuzumab or lapatinib in vivo in order to develop the optimal therapy for women with ErbB-2 positive breast cancer. Based on our findings, we propose a central hypothesis that ErbB-2 overexpression and activity de-regulates Jagged- 1 vesicular transport restricting Jagged-1-mediated Notch activation at the cell surface. Thus, resistance to anti-ErbB-2-targeted agents can be prevented and/or reversed by targeting Jagged-1 and/or Notch-1 directly. We have formulated this hypothesis based on our preliminary data suggesting that hyperactive ErbB-2 de- regulates vesicular trafficking of Jagged-1, and that Jagged-1 was required for trastuzumab-induced increase in Notch-1 transcriptional activity. To test our hypothesis and achieve the overall objective, we propose three Specific Aims: Specific Aim 1: Identify the molecular mechanism(s) responsible for Jagged-1-mediated Notch-1 activation in response to ErbB-2 inhibition. Our working hypothesis based on strong preliminary data is, that hyperactive ErbB-2 restricts proper Jagged-1 endocytosis to inhibit Notch activation at the cell surface. ErbB-2 inhibition rescues this effect, resulting in re-activation of Jagged-1-mediated Notch signaling. Specific Aim 2: Determine if Jagged-1 is the critical mediator of Notch Signalling to confer resistance to anti-ErbB-2 treatment in vitro. Our working hypothesis is that availability of cell surface Jagged-1 is critical to activating Notch-1 and thus promoting cell proliferation, survival, and resistance to anti-ErbB-2 treatment in vitro. Specific Aim 3: Evaluate the therapeutic efficacy of targeting Jagged-1 to prevent tumor recurrence and reverse resistance in vivo. Our working hypothesis is that simultaneous targeting of both ErbB-2 and Jagged-1-mediated Notch signaling pathways will prevent and/or reverse trastuzumab or lapatinib resistant ErbB-2 positive breast cancer in vivo providing pre-clinical evidence for a future prospective efficacy trial. Upon successful completion of these studies, we will begin a clinical trial to test our pre-clinical results. Our gal is development of scientifically-based evidence to support a therapeutic benefit of combining anti- ErbB-2 treatment with Notch pathway inhibition which provides a foundation for Phase I/Phase 2 clinical trials to test benefits in women with ErbB-2 positive breast cancer with the goal of preventing or reversing resistance. PUBLIC HEALTH RELEVANCE: Breast tumors that express high levels of ErbB-2 are currently treated with trastuzumab plus chemotherapy or lapatinib. This treatment has dramatically increased survival for women with ErbB-2 positive tumors. Unfortunately, these breast tumors over time become resistant to trastuzumab or lapatinib and most importantly, metastatic breast tumors are resistant even at the beginning of treatment. The major problem with resistance is eventual disease progression and ultimately death-associated with metastases to distant organs. Our goal is to determine how the Notch signaling pathway is regulated by ErbB-2 and thus determine the contribution of Notch signaling to resistance in ErbB-2 positive breast cancer cells. Most importantly, we will identify a new combination treatment using trastuzumab or lapatinib plus a Notch inhibitor to specifically prevent and/or reverse resistance in ErbB-2 positive breast cancer with the goal of extending lives.